Project 4-Abstract We approach the investigation of the biochemical and structural basis for the inhibitory immune response to FVIII, with a fresh perspective drawn from our extensive background in the biochemistry of the homologous and related coagulation protein, FV. We will, for the first time, systematically address the implication from human patient data that the antibody response to human FVIII differs fundamentally, in character and intensity, to that of human FV. Using comparable B domain deleted variants of both proteins injected into wild type mice, we propose to compare the total inhibitory antibody response. Further mapping of the differential response will be performed using chimeric variants of FVIII or FV containing individual domains from the human protein on the mouse background. Obvious differences will be further studied by examining the spectrum of the immune response to FV by hybridoma technology in comparison to that previously established with FVIII. A second hypothesis will investigate whether the differential response to the two proteins relates to mechanistic differences employed to maintain functional quiescence in each procofactor. Studies under Aim 2 further address whether the binding of FVIII to vWF plays a fundamental role in regulating the immune response to the cofactor using immunization strategies with FVIII mutants with variously impaired vWF binding. A second approach will test whether defined proteolytic derivatives of FVIII, normally produced during its life cycle, play a previously unconsidered role in interrogating the immune system and determining the inhibitory response to FVIII. Aim 3 draws on our structural advances with FV(a) derivatives to ask whether the structural framework for considering the effects of inhibitory antibody binding to FVIII is compromised by geometric constraints within the C domains imposed by crystal packing. We propose small angle scattering and high resolution Cryo- EM of FVIII in solution and bound to membrane nanodiscs to address whether conformational flexibility within the C domains underlies its solution conformation and when bound to membranes. We will use prototypic antibody inhibitors to C1 and C2 as well as the recombinant D?D3 fragment of vWF to test for the commonality of the idea that these ligands stabilize the C domains within FVIII in a sub-optimal for its high affinity interaction with membranes. Finally drawing from structural advances with FV(a) and FXa, we will use x-ray crystallography in combination with the other structural approaches to test the hypothesis that inhibitory antibodies to the A2 domain segregate into those that impact the binding of the cofactor to FIXa, to FX or both to some intermediate extent. Our proposed multi-pronged approach, within the larger context of this U54, will shed new light on basic details of the biochemistry underlying the inhibitor response to FVIII infusions in hemophilia A patients.